SUMMARY - Human GBPs as regulators of immunity to intracellular bacterial pathogens Guanylate binding proteins (GBPs) are host defense proteins that play diverse and critical roles in cell-autonomous immunity to intracellular bacterial pathogens. These proteins are major regulators of fundamental host defense modules and involved in processes such as inflammasome activation and the execution of direct bactericidal activities. However, almost all of this work has been performed in mouse models and antimicrobial functions of human GBPs (hGBPs) are still poorly characterized. In our recent studies we identified divergent host defense functions of the human GBPs and linked specific human GBPs to novel resistance mechanisms that protect against an important class of bacterial pathogens. Specifically, we found that human GBP1 (hGBP1) underpins two novel host defense mechanisms against cytosolic Gram-negative bacterial pathogens: 1) hGBP1 protein associates with cytosolic Gram- negative bacteria such as Shigella flexneri or Burkholderia thailandensis in the host cell cytosol. Once bound to bacteria, hGBP1 directly interferes with the actin-based intracellular motility of bacteria and thereby blocks bacterial dissemination; and 2) hGBP1 mediates activation of chemokine production by infected epithelial cells. Thus, hGBP1 activation appears to serve as a central node for two distinct but synergistic immune functions: a cell-intrinsic defense program prevents cell-to-cell spread of the pathogen while the production of paracrine immune signals promotes the activation and recruitment of immune cells to the site of infection. In this work, we will define the molecular mechanisms underlying these two important roles for hGBP1 that we have uncovered. In Aim1 we will combine cell culture and biochemical approaches to define the mechanism by which hGBP1 specifically detects Gram-negative bacteria in the host cell cytosol, a process critical for the inhibition of actin-based motility. In Aim2 we will characterize the mechanism by which hGBP1 bound to bacteria blocks actin-based motility. In Aim3 we will explore a second, independent function of hGBP1 as a novel regulator of an immune sensing pathway leading to the production of immune-modulatory chemokines using bacterial genetics, host genetics and cell biological approaches. In all of these studies we will take advantage of our recent discovery that the S. flexneri effector protein IpaH9.8 antagonizes hGBP1 function. Therefore, the use of IpaH9.8- deficient S. flexneri strains will enable us to monitor and functionally dissect an operational hGBP1-driven host response. Overall, the work proposed here will provide a fundamental understanding of the role of hGBP1 and other human GBPs in cell-autonomous immunity to intracellular bacterial pathogens. !